Genome sequence of the plant-growth-promoting bacterium Bacillus velezensis EU07

Abstract Many Gram-positive spore-forming rhizobacteria of the genus Bacillus show potential as biocontrol biopesticides that promise improved sustainability and ecological safety in agriculture. Here, we present a draft-quality genome sequence for Bacillus velezensis EU07, which shows growth-promotion in tomato plants and biocontrol against Fusarium head blight. We found that the genome of EU07 is almost identical to that of the commercially used strain QST713, but identified 46 single-nucleotide differences that distinguish these strains from each other. The availability of this genome sequence will facilitate future efforts to unravel the genetic and molecular basis for EU07's beneficial properties.


INTRODUCTION
Many Gram-positive spore-forming rhizobacteria of the genus Bacillus show potential as biocontrol biopesticides that promise improved sustainability and ecological safety in agriculture [1][2][3].Here, we present genomic sequencing data for Bacillus strain Egem-Utku 07, hereafter known as EU07.This strain was previously isolated from the rhizosphere of diseased tomato plants [4] in an effort to collect strains that could inhibit the soilborne pathogen Fusarium oxysporum f. sp.radicis-lycopersici [4], which causes crown rot in tomato.We demonstrated that EU07 inhibits this pathogen in vitro [4].Furthermore, EU07 promotes growth and inhibits fusarium head blight in planta [5].We previously established that EU07 is a member of the genus Bacillus, but its precise species identity was ambiguous.Furthermore, in the absence of sequence data, little was known about the potential molecular mechanisms for its beneficial properties.Here, we present a draft-quality genome sequence assembly and genomic sequence reads from strain EU07.This dataset will help in better understanding EU07's phylogeny and taxonomy, and provide a resource to assist elucidation of the molecular mechanisms of EU07's beneficial traits.

Bacterial strain and isolation of genomic DNA
We isolated genomic DNA from bacterial strain EU07 from fresh liquid culture grown for 24 h in nutrient broth pH 7.2.We note that this medium provides a laboratory environment quite different from the bacterium's normal soil environment.The liquid culture was inoculated from a single colony and, therefore, was assumed to be clonal.We used the ISOLATE II genomic DNA kit (Bioline), following the manufacturer's instructions.The quality and concentration of the genomic DNA were assessed using a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific).

DNA sequencing
Genomic DNA was sent to the University of Exeter's Sequencing Facility (https://biosciences.exeter.ac.uk/sequencing/) for Illumina Nextera XT library preparation and sequencing on the Illumina MiSeq platform to generate 748 528 pairs of 300 bp reads with a mean insert size of approximately 400 bp.

Genome sequence assembly
We performed adapter trimming and quality filtering on the MiSeq reads using Trim Galore version 0.6.7 [6], which incorporates Cutadapt version 3.5 [7].The -q parameter was set to 30 and we used the --paired option.The resulting cleaned read-pairs served as input for de novo assembly using SPAdes version 3.13.1 [8] with the --careful option.The resulting scaffolds and contigs were re-ordered against the reference genome of strain FZB42 with the Mauve Contig Mover [9].Annotation was added by the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline version 6.6 [10] after submission of the genome assembly.The command lines are documented in GitHub at https:// github.com/davidjstudholme/bacillus_EU07/tree/main/assemblyand in the Zenodo repository (https://doi.org/10.5281/zenodo.10968102)[11].

Assessment of genome-assembly quality
We calculated assembly statistics using quast version 5.2.0 [12].We checked read coverage of the genome assembly by aligning the EU07 reads against the EU07 assembly and calculating alignment statistics with Qualimap version 2.3 [13].The alignment was performed using bwa-mem version 0.7.17 [14]; then, we reformatted and sorted the output using SAMtools version 1.13 [15].The full details of the command lines are documented at https://github.com/davidjstudholme/bacillus_EU07/blob/main/assemblyQC/README.md and in the Zenodo repository [11].

Further whole-genome analyses
We used the Proksee web server [25] to perform several analyses of the assembled EU07 genome.This included blastn searches against 888 related genomes, annotation of horizontally acquired genomic regions with Alien Hunter [26], and identification of bacteriophage sequences using VirSorter [27,28] and Phigaro [29].Variant-calling was performed using the Parsnp tool in Harvest [30].

Genome sequencing and assembly
We generated 748 528 pairs of 300 bp Illumina MiSeq sequencing reads from EU07 genomic DNA.This represents approximately 100× coverage of the 4.2 Mbp genome.Trimming and filtering with Trim Galore left 715 442 pairs of reads, with lengths ranging from 20 to 300 bp.De novo assembly with SPAdes yielded 266 contigs with a total length of 4.2 Mbp and N 50 length of 52.8 kb.This was deposited in GenBank via the NCBI Submission Portal under accession number GCA_019997305.2.The NCBI's contamination filtering removed 5 contigs, leaving 261.The NCBI PGAP annotation system predicted 4 273 genes, of which 4 081 encode putative proteins.The results of NCBI's quality check with CheckM v1.2.2 [31,32] revealed a completeness of 98.16 % (85th percentile) and 0.47 % contamination.
Alignment of sequencing reads against the genome assembly and analysis with Qualimap revealed a mean coverage of 93.25× and standard deviation of 89.87.Almost all of the genome assembly (99.96 %) had at least 1× coverage, and 97.59 % of the assembly had at least 10× coverage.The full Qualimap report and output files are available in the Zenodo repository (https://doi.org/10.5281/zenodo.10968102)[11], allowing users of this data to take coverage into account when performing analyses.We note that the contig with least coverage is JAIFZJ020000237.1,having only 1.04× coverage.Nevertheless, blast searches reveal that this contig shows very high levels of sequence similarity to genomes of other Bacillus velezensis strains, increasing confidence in its validity.The phylogenomic maximum-likelihood tree was generated using PhaME and FastTree.The black star highlights the position of strain EU07, whose genome sequence is presented in this study.The configuration file and the tree files are deposited in GitHub at https://github.com/davidjstudholme/bacillus_EU07.Accession numbers for the genome assemblies can be found in Table 3.The tree can be viewed interactively at https://itol.embl.de/tree/14417323152242691702474608.Fig. 2. Whole-genome-sequence alignment between B. velezensis EU07 and closely related strains.Genome sequences were re-ordered, aligned and visualized using Mauve.Accession numbers for the genome assemblies can be found in Table 3. Green blocks in each genome are homologous to green blocks in all the other genomes.Blue blocks are homologous to the blue blocks in all the other genomes.

belongs to the species B. velezensis
Previously, the phylogenetic and taxonomic position of strain EU07 had been ambiguous and we previously referred to it as 'B.sp.' and 'B.subtilis' [4,5].To identify the species to which strain EU07 belongs, we uploaded the genome assembly to the Type Strain Genome Server (TYGS) [33].This classified EU07 to the species B. amyloliquefaciens.Among the sequenced type strains in TYGS, the most similar to EU07 was FZB42 [34], which is the type strain of B. amyloliquefaciens subsp.plantarum [35].However, this taxon is now considered to be synonymous with B. velezensis and distinct from B. amyloliquefaciens [36].
Hereafter, we refer to our strain as B. velezensis EU07.

EU07 belongs to a clade of plant-associated strains of B. velezensis
To identify previously sequenced similar genomes, we calculated ANI between B. velezensis EU07 and all 888 genome assemblies available in GenBank for the B. amyloliquefaciens group.This revealed that EU07 shares more than 99.9 % ANI with 13 previously sequenced genomes.Table 1 lists the genomes showing the highest levels of ANI to that of B. velezensis EU07.This includes strains that previously have been classified variously as B. amyloliquefaciens or B. velezensis.However, they all fall within the B. velezensis clade [36][37][38] and should be considered as belonging to that species.To further elucidate the evolutionary relationships of EU07, we generated a phylogenomic tree including these closely related strains and the relevant type strains; this is presented in Fig. 1.Consistent with the ANI results, strain EU07 falls within a clade that includes the same 13 strains that showed greatest ANI with EU07.Alignment of these genomes with Mauve (Fig. 2) reveals extensive conservation and co-linearity of the chromosome sequence among these strains.Comparison of the EU07 chromosome versus the genome sequences of related strains, as shown in Fig. 3, revealed that most of the presence-absence polymorphism was associated with loci predicted to originate from bacteriophage genomes.
Among the strains closely related to EU07 are several that previously have been described as having growth-promoting and/ or pathogen-inhibitory properties.For example, strain BS006 was isolated from roots of Physalis peruviana in Colombia and  promotes growth in banana [39].Strain KNU-28 was isolated from peach leaves in Korea [23].Strain ALB79 was isolated from grapes in northern California and shown to inhibit the growth of Listeria monocytogenes in vitro [40], while strain QST713 is used commercially (Serenade; Bayer) to protect mushroom crops against green mould disease and promotes growth in banana [37,41], among other applications.The endophytic Bacillus strain DMW1 was isolated from the inner tissues of potato tubers and exhibited strong biocontrol activity [42].The near-identity of these genome sequences, independently isolated from plants in diverse geographical locations, suggests that EU07 is a member of a widely disseminated lineage of B. velezensis with biocontrol and growth-promoting properties.The molecular mechanisms and genetic determinants of these properties have been extensively reviewed elsewhere [43][44][45], and include gene clusters for secondary metabolites such as bacilysin, fengycin and macrolactin, which are conserved in the B. velezensis lineage that includes BS006 and EU07 [38].
Since our previous phenotypic comparisons between strains EU07 and QST713 revealed differences in their abilities to suppress fungal growth, we compared their genome sequences to identify possible genetic determinants of the observed differences.Their genomes are almost identical, with no detectable differences in their gene contents.However, we identified 46 single-nucleotide differences, which are listed in Table 2.These differences appear to be non-uniformly distributed across the genome.For example, 20 of the 46 SNPs occur within a single gene that encodes the beta subunit of a class-1b ribonucleoside-diphosphate reductase [46] (RefSeq WP_108702400.1;locus tag BVQ_RS09140).This suggests that these differences might be explained by recombination events associated with horizontal genetic transfer rather than point mutations.We also identified some sequence differences between EU07 and QST713 in the intergenic regions between several tRNA genes (GenBank accession no.JAIFZJ010000168.1).These genetic differences may explain the previously observed differences observed between the DNA fingerprints of these two strains when previously assayed using RAPDs [4].

Conclusion
Genome sequencing of potential biocontrol strain EU07 revealed that it belongs to the species B. velezensis, a species often closely associated with plant roots, and well known for promoting plant growth and biocontrol.The EU07 strain is genetically almost identical to the commercially used strain QST713 (Serenade) and several other previously sequenced and characterized strains; however, we identified several genes containing single-nucleotide differences that can distinguish between EU07 and QST713.Strain EU07 is more distantly related to the commercially used B. velezensis strain FZB24 (TAEGRO), previously known as the type-strain of B. amyloliquefaciens subsp.plantarum.The availability of this genome sequence will facilitate future efforts to unravel the genetic and molecular basis for the strains beneficial properties.

Fig. 1 .
Fig. 1.Phylogenetic position of B. velezensis EU07 within the B. amyloliquefaciens group.The phylogenomic maximum-likelihood tree was generated using PhaME and FastTree.The black star highlights the position of strain EU07, whose genome sequence is presented in this study.The configuration file and the tree files are deposited in GitHub at https://github.com/davidjstudholme/bacillus_EU07.Accession numbers for the genome assemblies can be found in Table3.The tree can be viewed interactively at https://itol.embl.de/tree/14417323152242691702474608.

Table 1 .
Genomes that share more than 99 % ANI with B. velezensis EU07

Table 3 .
Genome sequences included in the phylogenomic analysis